Human monoclonal antibody specific for the f protein of respiratory syncytial virus (rsv)

ABSTRACT

This invention is directed to an antibody construct or fragment thereof derived from an RSV-infected human, such that the antibody construct binds with specificity to RSV fusion protein antigenic region II/A with an affinity of greater than 1×10 −9 M. Preferably, the antibody construct is capable of neutralizing RSV strains, including at least one RSV strain that is resistant to palivizumab. The invention further relates to nucleic acids encoding the antibody construct or portions thereof, and cell lines expressing the antibody. This invention further relates to methods for producing said antibody construct, and to the use of said anti-body construct for treating or preventing infection of a patient by RSV having a normal or mutated version of F protein.

FIELD OF THE INVENTION

This invention relates to an monoclonal antibody construct or antibody fragment specific for the fusion protein (F protein) of respiratory syncytial virus (RSV), nucleic acids encoding the antibody construct or fragment, and cell lines expressing the antibody construct or fragment. This invention further relates to methods for producing said monoclonal antibody construct or antibody fragment and to the use of said monoclonal antibody construct or antibody fragment for treating or preventing infection by RSV having a normal or mutated version of F protein.

DESCRIPTION OF THE RELATED ART

Respiratory syncytial virus (RSV) is one of the most common causes of respiratory infections in humans, and is the leading cause of infant hospitalization and a leading viral cause of death in infants. RSV epidemics recur annually during the winter season. The severity of outbreaks may vary from year to year due to co-circulation of 2 main RSV strains, group A and B. During a given annual epidemic, a large portion of the population develops RSV upper and/or lower respiratory tract infections.

Approximately two thirds of all infants are infected with RSV during their first year of life. Peak incidence of occurrence is observed at age 2-8 months; by 2 years of age 99% of children have been infected with RSV at least once, and 36% have had at least two infections. Most of these RSV infections cause minor upper respiratory illness and cold-like symptoms. Overall, 4 to 5 million children under 4 years of age acquire an RSV infection, and more than 120,000 children are hospitalized annually in the United States because of this infection.

Attempts at developing an effective vaccine have thus far been unsuccessful. Even upon repeated vaccinations, the human immune system is incapable of raising a sufficiently protective antibody-based immune response. Annually recurring infections are common even among previously infected individuals with normal immune functions, albeit few hospitalizations are seen among older children and adults with intact immune systems. The reasons for the absence of a protective immunity after vaccination or repeated infections are presently unknown. Currently, few treatment options are available for lower respiratory infections with RSV, and treatment must be initiated promptly at the onset of the infection to inhibit the replicating virus effectively. In 1986, the U.S. Food and Drug Administration (FDA) approved ribavirin, a broad-spectrum antiviral agent, for treatment of children with severe RSV disease.

F protein mediates fusion of the viral membrane with the target cell membrane and thus mediates virus genome entry into the target cell. F protein embedded into the membrane of infected cells mediates fusion between infected cells and their neighbors, leading to the syncytia formation characteristic of RSV infection. F protein is a type I fusion protein that rearranges from a metastable pre-fusion conformation to a highly stable post-fusion structure. This structural change in the protein is necessary for membrane fusion. Antibody binding to antigenic site II/A interferes with this structural change, thus preventing infection of the target cell as well as the fusion of infected cells with neighboring cells and the subsequent formation of syncytia.

Inhibition of the fusion mechanism prevents infection in vitro and in vivo, effectively neutralizing the virus. Previous experiments in animals indicated that protection against RSV infection is conferred mainly by neutralizing antibodies, in particular antibodies towards the F protein on the surface of the RSV particle. Rodent-derived monoclonal antibodies, such as MED-493 (also known as palivizumab or Synagis) were subsequently developed for use in humans at risk for RSV infections (Boeckh, M et al., 2001). Palivizumab was approved by the FDA in 1998 for use in high risk patients, and so far is the only monoclonal antibody directed against RSV that is approved for human use.

A detailed topological and operational map of epitopes involved in neutralization and fusion was constructed using rodent-derived neutralizing antibodies specific for F protein of RSV A2 strain. Three non-overlapping antigenic sites (A, B, and C) and one bridge site (AB) were identified. The commercially available anti-RSV monoclonal antibody, palivizumab, binds to a highly conserved region on the extracellular domain of mature F protein, referred to as antigenic site II or site A (antigenic site II/A), which is said to encompass amino acids 262 to 275.

The safety and efficacy of palivizumab validate the antigenic site II/A as a crucial and effective target for a monoclonal antibody for use as a prophylactic measure to prevent infections. Nonetheless, a subset of RSV strains are resistant to palivizumab, and use of palivizumab can select for such resistant strains.

A second-generation antibody, motavizumab, was generated by manipulating individual amino acids within the six complementarity determining regions (CDRs) of palivizumab Amino acids in these regions were individually substituted with any amino acid, and the best combinations for improved potency over palivizumab without changing specificity for the defined epitope were selected. Motavizumab is approximately ten-fold more potent than its predecessor, palivizumab, due to a higher affinity for the F protein. However, motavizumab does not provide significant improvement in protection against RSV infection in patients at high risk for contracting lower respiratory infections and has a higher risk of unwanted side effects, in particular allergic reactions. The FDA did not approve motavizumab for use in humans. As demonstrated by the clinical results, manipulation of a few amino acids in the sequence of an existing antibody can increase the risks for serious side effects in humans.

Further attempts have been made to improve on the palivizumab antibody. In RSV-Specific Binding Molecules and Means for Producing Them (U.S. Pat. No. 8,562,996) to Spits, a high affinity antibody against a different epitope region of RSV is presented. It appears the antibody may have a higher IC50 against certain RSV strains than palivizumab. In Human Antibodies to Respiratory Syncytial Virus F Protein and Methods of Use Thereof (U.S. Ser. No. 14/207797) by Gurnett, an antibody is disclosed with an epitope region overlapping that of palivizumab, with a higher affinity, and capable of neutralizing subtype A and B RSV. However, neither reference discloses any differences in the abilities of any antibodies to neutralize RSV isolates not neutralized by palivizumab.

Therefore, there exists a significant need for novel antibodies that target a broad range of RSV strains, including palivizumab-resistant strains. Furthermore, antibodies that have a lower risk of adverse reactions in humans are of interest.

SUMMARY OF THE INVENTION

This invention is predicated, in part, on the discovery that fully human antibody constructs that recognize the F protein of RSV are able to neutralize both palivizumab-resistant and palivizumab-sensitive RSV strains. Further, methods and compositions are presented to further enhance the pharmacokinetics and cytotoxicity of the antibodies.

All RSV strains that exhibit resistance to palivizumab have been shown to contain amino acid changes within a specific region in the antigenic region II/A on the F protein (Zhao, X et al., 2004). As an example, palivizumab-resistant RSV strains selected in vitro had mutations at position 272 of the fusion protein, from lysine to asparagine, methionine, threonine, glutamine, or glutamate. Variants containing mutations at positions 272 and 275 were detected in breakthrough patients.

The suitability of the F protein antigenic region II/A as a target for preventing RSV infection, including lower respiratory tract infection, has been confirmed by the efficacy of palivizumab. Although it was previously reported that most epitopes within the antigenic region II/A were constant among subgroup A viruses, several epitopes in site II/A are highly variable among subgroup B viruses. The antigenic site II/A on F protein, and in particular the epitope covered by palivizumab and motavizumab (amino acid positions in the region from 262 to 275) is subject to mutations at a frequency of 1 in 20 of all clinical isolates tested. Palivizumab is ineffective at preventing lower respiratory tract infections by these mutant strains. Thus, antibodies targeting the same antigenic region might exhibit improved efficacy as compared to palivizumab and motavizumab in cases where mutations are found in these amino acid positions. Therefore, an urgent need exists to develop novel monoclonal antibodies that bind to the antigenic region II/A of RSV F protein with higher affinity than palivizumab and that are capable of preventing and/or treating infection by palivizumab-resistant RSV strains.

Severe allergic reactions are common side effects of antibody-based preventive and/or therapeutic interventions, significantly limiting the use of such antibodies. Severe allergic side effects have been observed during the clinical testing of motavizumab. For example, an increased frequency of hypersensitivity reactions, including cases suggestive of anaphylaxis, was observed in clinical testing. Several patients treated with multiple doses of motavizumab developed anti-drug antibodies (ADA) and had severe allergic reactions due to ADA. Both palivizumab and motavizumab are mouse derived and not of human origin. Motavizumab's de novo-designed amino acid structure at the CDRs and its residual murine sequences are possible reasons for adverse reactions in late stage clinical studies, and motavizumab was ultimately deemed not safe for human use.

Antibodies generated by the human immune system against pathogens such as RSV inherently exhibit lower propensity to react with human self-antigens. In a preferred embodiment, the novel antibody constructs or antibody fragments targeting RSV described herein are entirely constructed from human origin.

Palivizumab is not sufficient to prevent infection by every RSV isolate. Therefore, a fully human, high-affinity antibody construct or antibody fragment with specificity to an RSV antigenic region (such as II/A) is highly desired. In one embodiment, the antibody construct or antibody fragment preferably recognizes both palivizumab-sensitive and palivizumab-resistant RSV strains. In one embodiment, the antibody construct or antibody fragment preferably has an affinity constant of greater than about 10⁻⁹ M, and preferably greater than about 10⁻¹⁰ M, or greater than about 10⁻¹¹ M. In one embodiment, the antibody construct or antibody fragment recognizes and neutralizes human RSV strains of type A and B. In one embodiment, the antibody construct or antibody fragment recognizes and neutralizes more than one RSV strain, and preferably one or more palivizumab-resistant RSV strains.

In one embodiment of the present invention, a fully human antibody construct or fragment thereof with specificity to RSV antigenic region II/A and an affinity constant of at least 10⁻⁹ M, and preferably greater than 10⁻¹⁰ M, is described. In another embodiment, the fully human antibody construct or fragment thereof is capable of neutralizing RSV virus strains of type A and B. In one embodiment, the antibody is capable of inhibiting binding by palivizumab. In one embodiment, the antibody construct or antibody fragment neutralizes one or more palivizumab-resistant strains.

In one embodiment, the invention relates to a cell comprising one or more cDNA sequences which encode a heavy chain variable region and/or a light chain variable region, wherein each cDNA sequence is constructed from an RSV-infected human, which cell produces an antibody construct or antibody fragment comprising the heavy chain variable region and/or the light chain variable region, such that the antibody construct or fragment thereof binds to RSV antigenic region II/A. In a preferred embodiment, the antibody construct or antibody fragment binds to RSV antigenic region II/A with an affinity of greater than 1×10⁻⁹. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a HEK293T cell. In one embodiment, the cell is a tobacco cell.

In one embodiment, the heavy chain variable region cDNA sequence is coupled to a cDNA sequence which encodes a constant region of human immunoglobulin. In a preferred embodiment, the constant region cDNA sequence is from a different patient than the heavy chain variable region and/or the light chain variable region. In one embodiment, the human immunoglobulin is from IgG1.

In one embodiment, the heavy chain variable region cDNA sequence comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one embodiment, the nucleotide sequence encoding a complementarity determining region (CDR)1 of the heavy chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 5. In one embodiment, the nucleotide sequence encoding CDR2 of the heavy chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 6. In one embodiment, the nucleotide sequence encoding CDR3 of the heavy chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 7.

In one embodiment, the light chain variable region cDNA sequence is coupled to a cDNA sequence which encodes a light chain constant region of human immunoglobulin. In a preferred embodiment, the constant region cDNA sequence is from a different patient than the light chain variable region and/or the heavy chain variable region. In one embodiment, the human immunoglobulin is from IgG1.

In one embodiment, the light chain variable region cDNA sequence comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the nucleotide sequence encoding CDR1 of the light chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 11. In one embodiment, the nucleotide sequence encoding CDR2 of the light chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 12. In one embodiment, the nucleotide sequence encoding CDR3 of the light chain of the antibody construct or antibody fragment comprises a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 13.

In one embodiment, this invention relates to a cDNA sequence as described herein. In one embodiment, this invention relates to an RNA molecule encoded by a cDNA sequence as described herein.

In one embodiment, this invention is directed to an antibody construct or antibody fragment that binds to RSV antigenic region II/A. In a preferred embodiment, the antibody construct or antibody fragment binds to RSV antigenic region II/A with an affinity of greater than 1×10⁻⁹ M. In one embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV strain. In a preferred embodiment, the antibody construct or antibody fragment is capable of neutralizing one or more RSV strains that are resistant to palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least 2 times greater than the neutralization capacity of palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least 3 times greater than the neutralization capacity of palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least 4 times greater than the neutralization capacity of palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least 5 times greater than the neutralization capacity of palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least 10 times greater than the neutralization capacity of palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least 15 times greater than the neutralization capacity of palivizumab.

In one embodiment, the heavy chain variable region of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the amino acid sequence of CDR1 of the heavy chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to GASINLYD (SEQ ID NO.: 8). In one embodiment, the amino acid sequence of CDR2 of the heavy chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to GYISGST (SEQ ID NO.: 9). In one embodiment, the amino acid sequence of CDR3 of the heavy chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to ARDVGWGPQYYYGLDV (SEQ ID NO.: 10).

In one embodiment, the light chain variable region of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the amino acid sequence of CDR1 of the light chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to HSVQSTS (SEQ ID NO.: 14). In one embodiment, the amino acid sequence of CDR2 of the light chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to GGS (SEQ ID NO.: 15). In one embodiment, the amino acid sequence of CDR3 of the light chain of the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to QQSDRSPPIT (SEQ ID NO.: 16).

In one embodiment, the antibody construct or antibody fragment recognizes an epitope on the F protein of RSV. In one embodiment, the antibody construct or antibody fragment recognizes an epitope within the antigenic region II/A of the RSV F protein. In one embodiment, the antibody construct or antibody fragment recognizes an amino acid sequence having at least 90% sequence homology to the epitope of SEQ ID NO.: 23 and/or SEQ ID NO.: 24.

In a preferred embodiment, the antibody construct or fragment is fully human. In one embodiment, the antibody construct or antibody fragment is chimeric. In one embodiment, the antibody construct or antibody fragment is humanized.

In one embodiment, the antibody construct or antibody fragment comprises (a) a heavy chain complementarity determining region (CDR)1 comprising the amino acid sequence GASINLYD (SEQ ID NO.: 8); (b) a heavy chain CDR2 comprising the amino acid sequence GYISGST (SEQ ID NO.: 9); (c) a heavy chain CDR3 comprising the amino acid sequence ARDVGWGPQYYYGLDV (SEQ ID NO.: 10); (d) a light chain CDR1 comprising the amino acid sequence HSVQSTS (SEQ ID NO.: 14), (e) a light chain CDR2 comprising the amino acid sequence GGS (SEQ ID NO.: 15), and (0 a light chain CDR3 comprising the amino acid sequence QQSDRSPPIT (SEQ ID NO.: 16).

In an aspect of the invention, the antibody or antibody fragment is constructed to comprise glycans lacking fucose and xylose, and/or incorporate an Fc region adapted to bind to neonatal receptor FcRn of epithelial cells with at least 10-fold (or 100-fold) less affinity than AR201. In some embodiments, the antibody is a modified version of AR201, e.g., retaining the 6 CDRs identified herein.

In one embodiment, this invention relates to a pharmaceutical composition comprising an antibody construct or antibody fragment as described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the antibody construct or antibody fragment is lyophilized. In one embodiment, the antibody construct or antibody fragment is in an aqueous solution. In one embodiment, this invention relates to a bag for intravenous therapy, comprising the antibody construct or antibody fragment as described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, this invention relates to a method of producing an antibody construct or functional part thereof, the method comprising culturing a cell as described above under conditions in which the cDNA sequences are expressed, thereby producing an antibody construct or fragment that binds with specificity to RSV antigenic region II/A. In a preferred embodiment, the antibody or a fragment thereof bind to RSV antigenic region II/A with an affinity of greater than 1×10⁻⁹M. In a further preferred embodiment, the antibody or fragment thereof is capable of neutralizing at least one RSV strain. In an especially preferred embodiment, the antibody or fragment thereof is capable of neutralizing one or more RSV strains that are resistant to palivizumab.

In one embodiment, this invention relates to a chromatography column or membrane comprising an antibody construct or antibody fragment as described herein, wherein the antibody construct or antibody fragment is bound to the chromatography column or membrane. In one embodiment, the chromatography column or membrane comprises Protein A, e.g. a Protein A resin. In one embodiment, the chromatography column or membrane comprises an ion exchange resin. In one embodiment, the chromatography column or membrane comprises a hydrophobic charge induction chromatography column.

In one aspect, this invention relates to a method for treating a patient infected with RSV by administering an antibody construct or antibody fragment as described herein to the patient. In one aspect, this invention relates to a method for preventing infection by RSV in a patient at risk for RSV infection by administering an antibody construct or antibody fragment as described herein to the patient. Administration may be by any suitable method, as determined by a skilled clinician. In a preferred embodiment, the antibody construct or antibody fragment is administered by intramuscular or intravenous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the process used to isolate an antibody specific for RSV F protein from selected and cultured human lymphocytes.

FIG. 2 describes the complete nucleotide sequence (SEQ ID NO.: 1) and the amino acid sequence (SEQ ID NO.: 2) of the variable region of the heavy chain of AR201.

FIG. 3 describes the complete nucleotide sequence (SEQ ID NO.: 3) and the amino acid sequence (SEQ ID NO.: 4) of the variable region of the kappa light chain of AR201.

FIG. 4 describes the complete nucleotide sequences (SEQ ID NO.: 5; SEQ ID NO.: 6; SEQ ID NO.: 7) and the amino acid sequences (SEQ ID NO.: 8; SEQ ID NO.: 9; SEQ ID NO.: 10) of the CDR regions of the variable region of the heavy chain of AR201.

FIG. 5 describes the complete nucleotide sequences (SEQ ID NO.: 11; SEQ ID NO.: 12; SEQ ID NO.: 13) and the amino acid sequences (SEQ ID NO.: 14; SEQ ID NO.: 15; SEQ ID NO.: 16) of the CDR regions of the variable region of the light chain of AR201.

FIG. 6 describes the binding of purified AR201 () and human non-specific monoclonal IgG1 (▴) to RSV-EIA antigen over a broad range of concentrations.

FIG. 7 describes the recognition and binding of reference strains of RSV-A and RSV-B by AR201.

FIGS. 8A and 8B compares the recognition of clinical isolates of RSV by AR201 (black bars) and palivizumab (white bars).

FIG. 8C indicates the ratio of binding of AR201 and palivizumab to clinical isolates of RSV. Black bars indicate a binding ratio of greater than 5 for the binding of AR201 over palivizumab to the respective clinical RSV isolate, indicative of resistance of the clinical isolate towards palivizumab.

FIG. 9A provides the nucleotide sequence (SEQ ID NO.: 17) of the F protein of a clinical isolate that is resistant to palivizumab.

FIG. 9B describes the amino acid sequence (SEQ ID NO.: 18) of the F protein of one clinical isolate resistant to palivizumab.

FIG. 10 provides the amino acid sequence of the palivizumab epitope region (SEQ ID NO.: 23), and the corresponding amino acid sequence of the F protein of a palivizumab-resistant strain (SEQ ID NO.: 24) that is recognized by AR201.

FIG. 11 provides the predicted peptides (SEQ ID NO.: 25 and SEQ ID NO.: 26) resulting from Asp-N digestion of RSV F protein that contain fragments of the palivizumab epitope region.

FIG. 12 describes Asp-N cleavage of RSV F protein that is bound by AR201 (black bars) or palivizumab (white bars).

FIG. 13 is a chart showing the relative prophylactic effectiveness of IM administered AR201 in protecting rats from an RSV challenge as compared to palivizumab.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of this invention will be limited only by the appended claims.

The detailed description of the invention is divided into various sections only for the reader's convenience and disclosure found in any section may be combined with that in another section. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of compounds.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein the following terms have the following meanings.

The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by (+) or (−) 10%, 5% or 1%.

“Comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

“Pharmaceutically acceptable composition” refers to a composition that is suitable for administration to a human. Such compositions include various excipients, diluents, carriers, and such other inactive agents well known to the skilled artisan.

“Therapeutically effective amount” or “therapeutic amount” refers to an amount of a drug or an agent that, when administered to a patient suffering from a condition, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of the condition in the patient. The therapeutically effective amount will vary depending upon the patient and the condition being treated, the weight and age of the subject, the severity of the condition, the salt, solvate, or derivative of the active drug portion chosen, the particular composition or excipient chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can be determined readily by one of ordinary skill in the art. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

“Treatment,” “treating,” and “treat” are defined as acting upon a disease, disorder, or condition with an agent to reduce or ameliorate harmful or any other undesired effects of the disease, disorder, or condition and/or its symptoms. “Treatment,” as used herein, covers the treatment of a patient, and includes: (a) reducing the risk of occurrence of the condition in a patient determined to be predisposed to the condition but not yet diagnosed as having the condition, (b) impeding the development of the condition, and/or (c) relieving the condition, i. e. , causing regression of the condition and/or relieving one or more symptoms of the condition. “Treating” or “treatment of” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results such as the reduction of symptoms. For purposes of this invention, beneficial or desired clinical results include, but are not limited to: preventing infection of a patient at risk of RSV infection; or reducing the severity of infection by RSV, e.g., by reducing one or more symptoms, reducing the length of time of infection, etc.

As used herein, the term “patient” refers to a mammal In a preferred embodiment, the patient is a human.

As used herein, the term “strain” or “RSV strain” refers to any RSV. Strains include, but are not limited to, clinical isolates, variants, mutants, and the like. Strains may be palivizumab-resistant or palivizumab-sensitive. Strains may be of either RSV type, A or B. general, strains are distinguishable by one or more genetic mutations, even if such mutation does not confer a different characteristic to the virus.

As used herein with regard to the binding of an antibody to an RSV peptide sequence, an “epitope region” is a sequence identified as including those amino acids that take part in the binding interaction with the antibody. For example, although certain assays have adequate resolution to identify a region of antigen amino acids within which antibody binding interactions take place, not all of the amino acids actually take part in the binding interaction. The “epitope”, as used herein, is the combination of amino acids within the epitope region that interact with the antibody to provide the specific binding between antigen and antibody.

As used herein, the term “antibody construct” refers to an antibody wherein at least a portion of the antibody is derived from an antibody from a human patient who had been exposed to the antigen of interest. An antibody construct may be an entire antibody or a fragment or portion thereof, provided the antibody, fragment, or portion has the recited affinity for F protein. An antibody construct may be fully human, humanized, or chimeric. An antibody construct may comprise amino acid sequences derived from a single patient, multiple patients, and/or known antibody sequences (e.g., a consensus constant region sequence).

As used herein, the term “antibody fragment” refers to any portion of the antibody that recognizes an epitope. Antibody fragments may be glycosylated. By way of non-limiting example, the antibody fragment may be a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment, a rIgG fragment, a functional antibody fragment, single chain recombinant forms of the foregoing, and the like. F(ab′)2, Fab, Fab′ and Fv are antigen-binding fragments that can be generated from the variable region of IgG and IgM. They vary in size, valency, and Fc content. The fragments may be generated by any method, including expression of the constituents (e.g., heavy and light chain portions) by a cell or cell line, or multiple cells or cell lines.

“F(ab′)2” fragments contain two antigen-binding regions joined at the hinge through disulfide linkages and lack most of the Fc region. “Fab′” fragments are derived from F(ab′)2 but include only one antigen binding region. They may contain a small portion of Fc.

“Fab” fragments are monovalent fragments produced from IgG and IgM, consisting of the variable heavy chain, constant heavy chain, and variable light chain, constant light chain regions, linked by an intramolecular disulfide bond.

“Fv” is the smallest fragment produced from IgG and IgM that contains a complete antigen-binding site. Fv comprises a portion of the variable heavy and light chains, held together by non-covalent interactions. These chains tend to dissociate upon dilution, but can be cross-linked, for example using glutaraldehyde, intermolecular disulfides or a peptide linker. Fv fragments have the same binding properties and similar three-dimensional binding characteristics as Fab.

“rIgG” refers to reduced IgG or half-IgG, containing one heavy chain and one light chain. rIgG can be produced by selectively reducing the hinge-region disulfide bonds.

As used herein, the term “fully human antibody” refers to an antibody, antibody construct, or antibody fragment consisting entirely of human amino acid sequence. That is, the amino acid sequence of the human monoclonal antibody construct is derived from a human cell. This may be obtained in different ways. For example, the human monoclonal antibody construct consisting of human amino acid sequence can be obtained from a cell engineered to express the variable region heavy and light chains and/or CDRs from an antibody derived from a human patient (e.g., a patient who had been exposed to RSV and/or RSV F protein). Alternatively, the human monoclonal antibody construct can be obtained from a hybridoma, wherein the B-cell is a human B-cell. Alternatively, the human monoclonal antibody construct may be obtained by CDR grafting of the CDR regions, for example those indicated in FIGS. 4 and 5, onto available human monoclonal antibodies, thereby producing a human monoclonal antibody construct in accordance with the present invention. The entirely human amino acid sequence of the human monoclonal antibody construct prevents the occurrence of undesired adverse effects such as rejection reactions or anaphylactic shock.

The term “neutralizing” or “neutralizing capacity” as used herein refers to the ability of the antibody construct to attenuate infectivity by the virus. For example, the antibody construct may render the viral fusion protein ineffective such that the fusion between the virus and a cell, and/or between infected cells, is blocked or attenuated. An antibody having at least twice the neutralizing capacity of palivizumab is set forth herein, as shown in Example 6 (as determined by infectivity assay).

Monoclonal Antibody Constructs and Fragments Thereof

The current invention relates to a monoclonal antibody construct or antibody fragment that specifically bind the RSV F protein. Preferably, the antibody construct or antibody fragment specifically binds to the F protein antigenic region II/A.

In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of greater than 1×10⁻⁹ M. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of greater than 1×10⁻¹⁰ M. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of greater than 5×10⁻¹⁰ M. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of greater than 1×10⁻¹¹ M. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of greater than 1×10⁻¹² M. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of greater than 5×10⁻¹² M.

In one embodiment, the antibody construct or antibody fragment binds to F protein with a higher affinity than palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of between about 2:1 and about 500:1 versus palivizumab. In a preferred embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of between about 20:1 and about 200:1 versus palivizumab. It is to be understood that the ratio can be ranges between any two of these values, or any value there between (including endpoints). In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 2:1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 5:1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 10:1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 50:1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 100:1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 200:1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 300:1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 400:1 versus palivizumab. In one embodiment, the antibody construct or antibody fragment binds to F protein with an affinity of at least about 500:1 versus palivizumab.

In one aspect, the antibody construct or antibody fragment is capable of neutralizing RSV virus strains. In one embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV strain. In one embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV type A strain. In one embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV type B strain. In a preferred embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV type A strain and at least one type B strain. In an especially preferred embodiment, the antibody construct or antibody fragment is capable of neutralizing one or more strains that are resistant to palivizumab.

In one embodiment, the antibody construct or antibody fragment is capable of neutralizing at least one RSV strain with a greater neutralization capacity than palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least two times greater than the neutralization capacity of palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least five times greater than the neutralization capacity of palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least ten times greater than the neutralization capacity of palivizumab. In one embodiment, the neutralization capacity of the antibody construct or antibody fragment against at least one RSV strain is at least fifteen times greater than the neutralization capacity of palivizumab.

In one aspect, the antibody construct or antibody fragment comprises an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 96% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 97% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 98% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 99% sequence homology to the amino acid sequence of SEQ ID NO.: 1. In one embodiment, the antibody construct or antibody fragment comprises the amino acid sequence of SEQ ID NO.: 1. In a preferred embodiment, the heavy chain variable region of the antibody construct or antibody fragment comprises the amino acid sequence of SEQ ID NO.: 1.

In one aspect, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 96% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 97% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 98% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 99% sequence homology to the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with the amino acid sequence of SEQ ID NO.: 8, SEQ ID NO.: 9, and/or SEQ ID NO.: 10. In a preferred embodiment, the antibody construct or antibody fragment comprises a heavy chain CDR1 with the amino acid sequence of SEQ ID NO.: 8, a heavy chain CDR2 with the amino acid sequence of SEQ ID NO.: 9, and a heavy chain CDR3 with the amino acid sequence of SEQ ID NO.: 10.

In one aspect, the antibody construct or antibody fragment comprises an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 96% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 97% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 98% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises an amino acid sequence having at least 99% sequence homology to the amino acid sequence of SEQ ID NO.: 3. In one embodiment, the antibody construct or antibody fragment comprises the amino acid sequence of SEQ ID NO.: 3. In a preferred embodiment, the light chain variable region of the antibody construct or antibody fragment comprises the amino acid sequence of SEQ ID NO.: 3.

In one aspect, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16 In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 90% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 95% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16 . In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 96% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 97% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 98% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with an amino acid sequence having at least 99% sequence homology to the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In one embodiment, the antibody construct or antibody fragment comprises at least one CDR with the amino acid sequence of SEQ ID NO.: 14, SEQ ID NO.: 15, and/or SEQ ID NO.: 16. In a preferred embodiment, the antibody construct or antibody fragment comprises a light chain CDR1 with the amino acid sequence of SEQ ID NO.: 14, a light chain CDR2 with the amino acid sequence of SEQ ID NO.: 15, and a light chain CDR3 with the amino acid sequence of SEQ ID NO.: 16.

The invention further relates to derivatives of the antibody construct or antibody fragment described herein. The term “derivative” encompasses any mutants of the antibody construct differing by the addition, deletion, and/or substitution of at least one amino acid. Preferably, the derivative is a mutant of the antibody construct that carries at least one conservative substitution in any of the CDRs in the heavy chain and/or light chain as indicated in FIGS. 4 and 5. More preferably, the mutant has not more than 5, not more than 4, preferably not more than 3, particularly preferred not more than 2 conservative substitutions. The capacity of the fragment or derivative of the antibody to bind to the epitope can be determined by direct ELISA, for example, as described in the Examples section below.

In accordance with the present invention, the term “conservative substitution” means a replacement of one amino acid belonging to a particular physico-chemical group with an amino acid belonging to the same physico-chemical group. The physico-chemical groups are defined as follows: The group of non-polar amino acids comprises: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan. The group of amino acids having uncharged polar side chains comprises asparagine, glutamine, tyrosine, cysteine, and cysteine. The physico-chemical group of amino acids having a positively charged polar side chain comprises lysine, arginine, and histidine. The physico-chemical group of amino acids having a negatively charged polar side chain comprises aspartic acid and glutamic acid, also referred to as aspartate and glutamate.

In one embodiment, the light chain of the antibody construct or antibody fragment according to the present invention is of the kappa or lambda type. In a preferred embodiment, the light chain is of the kappa type. The light chain may be either a naturally occurring chain including a naturally rearranged, a genetically modified or synthetic type of light chain. According to a further embodiment, the heavy chain of the antibody of the present invention is selected from all human isotypes, namely IgM, IgA, or IgG. The light chain and heavy chain may either be covalently linked as a single-chain antibody (e. g. bivalent scFv, bifunctional scFv and bispecific scFv) or non-covalently linked with each other.

In one aspect, the antibody construct or antibody fragment recognizes an epitope on the F protein of RSV. In one embodiment, the epitope is within antigenic region II/A. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 85% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 90% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 95% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least sequence 96% homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 97% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 98% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 99% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 23 or SEQ ID NO.: 24.

In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 85% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 90% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 95% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least sequence 96% homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 97% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 98% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 25 or SEQ ID NO.: 26. In one embodiment, the antibody construct recognizes an epitope within antigenic region II/A having at least 99% sequence homology to a portion of the amino acid sequence of SEQ ID NO.: 26 or SEQ ID NO.: 26.

The present invention further relates to nucleotide sequences encoding the antibody construct or antibody fragment, or portions thereof. In one embodiment, the nucleotide sequence comprises a cDNA sequence. In one embodiment, the nucleotide sequence comprises a cDNA sequence encoding for the heavy chain variable region. In one embodiment, the cDNA sequence encoding for the heavy chain variable region has at least 85% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one embodiment, the cDNA sequence encoding for the heavy chain variable region has at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one embodiment, the cDNA sequence encoding for the heavy chain variable region has at least 95% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one embodiment, the cDNA sequence encoding for the heavy chain variable region has at least 96% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one embodiment, the cDNA sequence encoding for the heavy chain variable region has at least 97% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one embodiment, the cDNA sequence encoding for the heavy chain variable region has at least 98% sequence homology to the nucleotide sequence of SEQ ID NO.: 2. In one embodiment, the cDNA sequence encoding for the heavy chain variable region has at least 99% sequence homology to the nucleotide sequence of SEQ ID NO.: 2.

In one embodiment, the nucleotide sequence comprises a cDNA sequence encoding for the light chain variable region. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 85% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 95% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 96% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 97% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 98% sequence homology to the nucleotide sequence of SEQ ID NO.: 4. In one embodiment, the cDNA sequence encoding for the light chain variable region has at least 99% sequence homology to the nucleotide sequence of SEQ ID NO.: 4.

In one embodiment, the nucleotide sequence encodes for one or more CDRs. In one embodiment, the nucleotide sequence encoding for one or more CDRs comprises a nucleotide with at least 85% sequence homology to the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13. In one embodiment, the nucleotide sequence encoding for one or more CDRs comprises a nucleotide with at least 90% sequence homology to the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13. In one embodiment, the nucleotide sequence encoding for one or more CDRs comprises a nucleotide with at least 96%, 97%, 98%, or 99% sequence homology to the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13. In a preferred embodiment, the nucleotide sequence encoding for one or more CDRs comprises the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13. In an especially preferred embodiment, the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, and/or SEQ ID NO.: 7 encodes for a CDR of the heavy chain variable region of the antibody construct or antibody fragment. In one embodiment, the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, and/or SEQ ID NO.: 7 encodes for a CDR of the light chain variable region of the antibody construct or antibody fragment. In one embodiment, the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13 encodes for a CDR of the heavy chain variable region of the antibody construct or antibody fragment. In an especially preferred embodiment, the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13 encodes for a CDR of the light chain variable region of the antibody construct or antibody fragment.

In one embodiment, the nucleotide sequence is a cDNA sequence. In one embodiment, the nucleotide sequence is an RNA molecule encoded by a nucleotide comprising SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 11, SEQ ID NO.: 12, and/or SEQ ID NO.: 13. In one embodiment, the nucleotide sequence is an RNA molecule encoded by a nucleotide comprising SEQ ID NO.: 2. In one embodiment, the nucleotide sequence is an RNA molecule encoded by a nucleotide comprising SEQ ID NO.: 4.

The present invention further provides vectors comprising at least one nucleic acid encoding the light chain of the antibody construct or antibody fragment of the present invention and/or at least one nucleic acid encoding the heavy chain of the antibody construct or antibody fragment of the present invention. The nucleic acids may be either present in the same vector or may be present in the form of binary vectors. The vector preferably comprises the promoter operatively linked to the nucleic acid in order to facilitate expression of the nucleic acid encoding the light and/or heavy chain. Preferably, the vector also includes an origin for replication and maintenance in a host cell. The vector may also comprise a nucleotide sequence encoding a signal sequence located 5′ of the nucleic acid encoding the light chain and/or heavy chain. The signal sequence may facilitate secretion of the encoded chain into the medium. The vector may further comprise a His-tag coding nucleotide sequence resulting in the expression of a construct for producing a fusion product with a His-tag at the N-terminus of the light and/or heavy chain of the antibody construct or antibody fragment, which facilitates purification of the protein.

In one embodiment, the antibody construct or antibody fragment according to the present invention is modified. The modifications include the di-, oligo-, or polymerization of the monomeric form e. g. by cross-linking using dicyclohexylcarbodiimide. The di-, oligo-, or polymers can be separated from each other by gel filtration. Further modifications include side chain modifications, e. g. modifications of ε-amino-lysine residues, or amino and carboxy-terminal modifications, respectively. Further modifications include post-translational modifications, e.g. glycosylation and/or partial or complete deglycosylation of the protein, and disulfide bond formation. The antibody construct or fragment may also be conjugated to a label, such as an enzymatic, fluorescent or radioactive label.

In one embodiment, the antibody construct or antibody fragment is a chimeric antibody. In one embodiment, the antibody construct or antibody fragment is a humanized antibody. In a preferred embodiment, the antibody construct or antibody fragment is a human antibody. In an especially preferred embodiment, the antibody construct or antibody fragment is a fully human antibody.

In one embodiment, the antibody construct or antibody fragment is lyophilized.

In one aspect, this invention relates to a pharmaceutical composition comprising the antibody construct or antibody fragment as described herein. Such compositions include various excipients, diluents, carriers, and such other inactive agents well known to the skilled artisan. In one embodiment, the pharmaceutical composition comprises the antibody construct or antibody fragment and a pharmaceutically acceptable carrier, diluent, or excipient.

Methods of Making Antibody Constructs and Fragments Thereof

This invention further relates to methods and composition for making the antibody construct or antibody fragment as described herein.

In one embodiment, human B-cells are obtained from patients who have been exposed to RSV and/or RSV F protein (e.g., convalescing patients or patients immunized with F protein or a fragment thereof). Blood samples can be taken from the patients and human B-cells can be isolated in a known manner (e.g., Current Protocols in Immunology. Chapter 7.1. Isolation of whole mononuclear cells from peripheral blood and cord blood. Published by Wiley & Sons, Eds: J C Coligan et al.). In one embodiment, the human B-cell may be fused to a myeloma or heteromyeloma to produce a hybridoma in accordance with known techniques according to the classical Kohler and Milstein approach, as described by Lang et al “Prophylaxis and therapy of Paseudomonas aeruginosa infection in cystic fibrosis and immunocompromised patients” Vaccine 22: S44-S48 (2004), which is incorporated herein by reference in its entirety. In a preferred embodiment, the B cell is cultured and the cDNA sequence of a heavy chain variable region, light chain variable region, and/or one or more CDRs is isolated therefrom. The cDNA sequences can be used to generate one or more vectors. Methods of producing fully human antibodies are described, for example, in Beerli et al. “Isolation of Human Monoclonal Antibodies by Mammalian Cell Display,” PNAS 105(38): 14336-14341 (2008) each of which is incorporated herein by reference in its entirety.

The vector(s) can be introduced into a cell such that the cell produces the antibody construct, antibody fragment, or portion thereof. The cell may be a prokaryotic cell or a eukaryotic cell. In a preferred embodiment, the cell is a plant cell or a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a HEK293 cell. In one embodiment, the cell is a PerC6 cell, a CHO cell, a COS cell, or a HELA cell.

In one embodiment, the antibody construct or antibody fragment described herein is produced in a plant cell. Methods of producing antibodies in plant cells are described, for example, in U.S. Pat. Nos. 8,119,406 and 8,513,397, each of which is incorporated herein by reference in its entirety. In a preferred embodiment, the cell is from a tobacco plant (e.g., genus Nicotiana). Benefits can be realized from expression in a plant host system. For example, the alternate glycosylations of a plant system can improve pharmacokinetics and/or desirable cytotoxic effects of the product antibody.

Preferably, the host cell comprises at least one nucleic acid encoding the light chain and at least one nucleic acid encoding the heavy chain and is capable of assembling the antibody construct such that a 3-dimensional structure is generated which is equivalent to the 3-dimensional structure of a human antibody produced by a human B-cell. If the light chain is produced separately from the heavy chain, then both chains may be purified and subsequently be assembled to produce an antibody having essentially the 3-dimensional structure of a human antibody as produced by a human B-cell. Alternatively, the host cell comprises at least one nucleic acid encoding at least the antigen-binding portion of the light chain and at least one nucleic acid encoding at least the antigen-binding portion of the heavy chain, and is capable of assembling the antibody construct or fragment such that the antibody construct or fragment is capable of binding the antigen.

The antibody construct or antibody fragment may also be obtained by recombinant expression of the encoded light and/or heavy chain (or portion thereof), wherein the nucleic acid is produced by isolating a nucleic acid encoding a human antibody and grafting of the nucleic acid sequence encoding the CDRs as defined in the figures onto the isolated nucleic acid.

Antibodies or antibody fragments can be purified, for example from cell culture supernatant, by any method. Exemplary methods of antibody purification are described in Liu et al., “Recovery and purification process development for monoclonal antibody production,” mAbs 2(5): 480-499 (2010), which is incorporated herein by reference in its entirety.

In one aspect, this invention relates to a chromatography column or membrane comprising an antibody construct or antibody fragment as described herein, wherein the antibody construct or antibody fragment is bound to the chromatography column or membrane. In one embodiment, the chromatography column or membrane comprises Protein A, e.g. a Protein A resin. In one embodiment, the chromatography column or membrane comprises an ion exchange resin. In one embodiment, the chromatography column or membrane comprises a hydrophobic charge induction chromatography column.

Although the descriptions and examples herein are directed to production of the antibody construct or fragment within a single cell or cell line, it is contemplated that one or more portions of the antibody construct or fragment may be produced by separate cells or cell lines and then combined to form a functional antibody construct or fragment. For example, the heavy chain (or portion thereof) may be produced by one cell, and the light chain (or portion thereof) may be produced by a second cell. The components may be isolated or purified, e.g. from the cell culture supernatant, and covalently or non-covalently linked by routine methods.

In one embodiment, this invention relates to a method for determining the potency of a putative antibody to strains of RSV that are resistant to commercially available antibodies, e.g., palivizumab. The binding affinity, neutralizing capacity, antigenicity, or any other measure of the usefulness of the putative antibody against at least one RSV strain may be compared to the antibody construct of the current invention. In one embodiment, a putative antibody that is determined to bind with an affinity equal to or greater than, have a neutralization capacity equal to or greater than, and/or exhibit equal or less antigenicity than, an antibody construct as described herein is further tested for effectiveness against RSV.

Methods of Treatment

This invention further relates to methods of treatment using the antibody construct or antibody fragment as described herein.

In one aspect, this invention relates to methods of preventing RSV infection in a patient at risk of infection by RSV. In one embodiment, upper respiratory infection is prevented. In one aspect, lower respiratory infection is prevented. In one aspect, both upper and lower respiratory infection is prevented.

In one aspect, this invention relates to methods of treating RSV infection in a patient. In one embodiment, upper respiratory infection is treated. In one aspect, lower respiratory infection is treated. In one aspect, both upper and lower respiratory infection is treated.

In one embodiment, the patient is a human. In a preferred embodiment, the patient is an infant (e.g., under 12 months of age). In one embodiment, the patient is a human having a condition that increases the risk of RSV infection. Conditions that increase the risk of RSV infection include, but are not limited to, being under 6 months of age; being under 12 months of age and born prematurely (e.g., before 40 weeks gestational age); lung disease; chronic obstructive pulmonary disease; congenital heart disease; congestive heart failure; weakened immune system; asthma; immunodeficiency (e.g., transplanted organ, leukemia, HIV/AIDS). While RSV infections are considered problematic mainly for (preterm) infants, also elderly and immune-compromised patients (e.g. HIV positive patients, patients under chemotherapy or patients with another severe underlying condition) are at risk. Palivizumab is not normally used to treat these patient groups due to low efficacy. AR-201 shows higher affinity for the virus, recognizes more viral strains and is currently being engineered for enhanced ADCC and prolonged half-life. Such improved potency and durability of action are expected to enhance the medicinal value of an anti-RSV mAb in immune-compromised and elderly patients infected with RSV.

In one embodiment, the antibody construct or antibody fragment is administered to a patient at risk of infection by RSV. In one embodiment, the antibody construct or antibody fragment is administered to a patient having or believed to have an infection by RSV. In one embodiment, a therapeutically effective amount of the antibody construct or antibody fragment is administered. In one embodiment, the therapeutically effective amount is between about 1 mg per kg body weight and about 1000 mg per kg body weight. In a preferred embodiment, the therapeutically effective amount is between about 1 mg per kg body weight and about 100 mg per kg body weight. In a more preferred embodiment, the therapeutically effective amount is between about 5 mg per kg body weight and about 50 mg per kg body weight. It is to be understood that the amount can be a range between any two of these values, or any value there between (including endpoints).

The antibody construct or antibody fragment may be administered by any appropriate route. The compositions, provided herein or known, suitable for administration in accordance with the methods provide herein, can be suitable for a variety of delivery modes including, without limitation, intramuscular and intravenous delivery. Compositions suitable for internal, pulmonary, rectal, nasal, vaginal, lingual, intraarterial, intraperitoneal, intracutaneous and subcutaneous routes may also be used. Sustained release dosage forms may also be used. All dosage forms may be prepared using methods that are standard in the art (see e.g., Remington's Pharmaceutical Sciences, 16th ed., A. Oslo editor, Easton Pa. 1980).

In one embodiment, the antibody construct or fragment is administered once. In a preferred embodiment, the antibody construct or fragment is administered multiple times. In a more preferred embodiment, the antibody construct or fragment is administered once a month during RSV season. Most preferably the antibody construct or fragment is administered only once per season. RSV season is generally November through April (in the Northern Hemisphere), but can be longer depending on the area and other factors (e.g., the primary strains that are circulating in a given year).

In one embodiment, the invention relates to a bag for intravenous delivery, comprising an IV bag containing the antibody construct or antibody fragment and a pharmaceutically acceptable excipient.

EXAMPLES

Unless stated otherwise, the abbreviations used throughout the specification have the following meanings:

-   -   cDNA=complimentary deoxyribonucleic acid     -   CDR=complementarity determining region     -   ELISA=enzyme-linked immunosorbent assay     -   FCS=fetal calf serum     -   g=gram     -   HC=heavy chain     -   HRP=horseradish peroxidase     -   Ig=immunoglobulin     -   KD=dissociation constant     -   kg=kilogram     -   LC=light chain     -   M=molar     -   mAb=monoclonal antibody     -   mg=milligram     -   mM=minute     -   mL=milliliter     -   mM=millimolar     -   ng nanogram     -   nM=nanomolar     -   PBS=phosphate buffer saline     -   PCR=polymerase chain reaction     -   pM=picomolar     -   RNA=ribonucleic acid     -   RSV=respiratory syncytial virus     -   RT-PCR=reverse transcription PCR     -   μg=microgram     -   μL=microliter     -   μM=micromolar     -   ° C.=degree Celsius

These one-letter symbols have the following meaning when representing amino acids:

-   -   A=Alanine     -   R=Arginine     -   N=Asparagine     -   D=Aspartic acid     -   C=Cysteine     -   E=Glutamic acid     -   Q=Glutamine     -   G=Glycine     -   H=Histidine     -   I=Isoleucine     -   L=Leucine     -   K=Lysine     -   M=Methionine     -   F=Phenylalanine     -   P=Proline     -   S=Serine     -   T=Threonine     -   W=Tryptophan     -   Y=Tyrosine     -   V=Valine

When representing nucleic acids, A=Adenine; T=Thymine; C=Cytosine; G=Guanine; U=Uracil, N=any nucleic acid.

The following Examples are intended to further illustrate certain embodiments of the disclosure and are not intended to limit its scope.

Example 1 Selection of Human B-cells Specific for RSV

For the generation of an antibody construct specific for the RSV F protein, human lymphocytes from recently resected tonsils of RSV infected human infants were isolated by mechanical disruption and passaging through a cell strainer. Isolated lymphocytes were depleted in parallel from monocytes by plastic adhesion in cell culture flasks, and subsequently RSV specific B cells were enriched by incubation of lymphocytes with immobilized total RSV antigen. For this purpose, 6-well plates were coated with total RSV antigen (EIA-antigen) at 10 μg/mL in phosphate buffer saline (PBS) overnight. After coating, the wells were blocked by incubation with 10% fetal calf serum (FCS) in PBS. Between one and ten million monocyte-depleted cells were incubated per one well of the coated six-well plate. Following incubation for one hour, the wells were washed and bound cells were harvested by trypsinization. The cells were added to plates containing cell culture medium containing 10% FCS and 10% conditioned supernatant with 20,000 EL-4B5 feeder cells (kind gift from the Geneva University Hospital, Geneva, Switzerland) that had been irradiated at 5000 cGy by a Gammacell 40 Research Irradiator. Conditioned supernatant was generated by stimulating isolated peripheral blood lymphocytes with phytohaemagglutinin (PHA, 5 μg/mL) and phorbol myristate acetate (PMA, 10 ng/mL) for 36 hours, followed by removal of cells and debris prior to cryopreservation. The conditioned supernatant was thawed prior to addition to the RSV-EIA selected lymphocytes. After up to ten days, small aliquots of the cell culture supernatants were analyzed for specific antibodies by RSV EIA ELISA.

ELISA plates were coated with RSV-EIA antigen or purified RSV F protein (Human RSV (A2) Fusion glycoprotein RSV F protein, Sino Biological Inc., Beijing, China) at 0.5 pg/mL overnight at 4 ° C., then blocked with 0.5% BSA in PBS. Cell culture supernatants were diluted with PBS containing 0.05% Tween (PBS-Tween) and aliquoted into wells. After incubation and subsequent washing with PBS-Tween, horseradish peroxidase- (HRP) labeled goat anti-human IgG was used to detect human IgG bound to the antigen. Positive wells were identified by colorimetric measurement, and cells from positive wells were expanded at low density in the presence of irradiated feeder cells and cell culture medium containing 10% FCS and 10% conditioned supernatant (as described above) over a period of several days. After retesting supernatants for specificity to RSV F protein, cells from positive wells were collected and processed for RNA isolation.

Example 2 Generation of Human Monoclonal Antibody to RSV

The method for isolating the specific antibody is summarized in FIG. 1. Specifically, RNA from selected B-cells was used to generate 5′RACE cDNA, followed by isotype specific RT-PCR to identify the variable region of the heavy chain (HC) and light chain (LC) as described by Welschof M, et al., 1995. The variable region of the HC was combined by PCR with the constant region of an IgG1 which is essentially in accordance with the IMGT reference sequence Y14737 (Lefranc, M.-P. et al., 2001, The Immunoglobulin Facts Book Academic Press, London, UK) and cloned into the eukaryotic expression vector pcDNA3.3-Topo (Invitrogen, USA). The whole coding region of the LC was amplified by RT-PCR and cloned into the expression vector. Subsequently, the vectors were transiently transfected into HEK293T cells [ATCC # CRL-11268 (American Type Culture Collection (ATCC), Manassas, Va. 20110 USA)]. Four to five days after transfection, supernatants were tested by ELISA for presence of antigen-binding antibodies as described above. The most promising candidate, AR201 (also called KBRV201), was selected for sequencing of the variable regions of the heavy and light chains. The final vectors were amplified and analyzed by Sanger sequencing.

The nucleotide and corresponding amino acid sequences are provided in FIGS. 2 (heavy chain, SEQ ID NO.: 1 and 2) and 3 (light chain, SEQ ID NO.: 3 and 4). The nucleotide and corresponding amino acid sequences of individual CDR regions are provided in FIGS. 4 (heavy chain: CDR1, SEQ ID NO.: 5 and 8; CDR2, SEQ ID NO.: 6 and 9; CDR3, SEQ ID NO.: 7 and 10) and 5 (light chain: CDR1, SEQ ID NO.: 11 and 14; CDR2, SEQ ID NO.: 12 and 15; CDR3, SEQ ID NO.: 13 and 16).

Example 3 Binding of Human Monoclonal Antibody to RSV F Protein Antigen

The isolated and sequenced human antibody AR201 was tested for binding characteristics to RSV F protein, as shown in FIG. 6. Hek293T cells were seeded at 300,000 cells per well of a six-well plate and cultivated overnight. After one day, medium was exchanged, and a freshly prepared solution of expression plasmids was added carefully to the cells. Cells were incubated for another 24 hours and medium was exchanged again for Pro293a-CDM medium. After three to five days, the resulting supernatant containing recombinant antibody was collected and antibody was purified by affinity chromatography with a HiTrap Protein A sepharose affinity column (GE Healthcare Life Sciences, Pittsburgh, Pa., USA). Resulting antibody was stored at −20 ° C. until use. For binding experiments, ELISA plates were coated as described above, and serial dilutions of purified AR201 or an unrelated fully human monoclonal IgG1 antibody (control) were added. After careful washing, bound antibodies were detected with anti-human IgG-HRP labeled secondary antibody. EC50 values were calculated based on colorimetric measurement of the bound antibody applying a variable slope-four parameter equation (GraphPad Prism Software V5.02, GraphPad Software Inc. San Diego, Calif., US). The EC50 value of AR201 was calculated as 0.1031 nM. No EC50 value could be calculated for the control monoclonal human IgG1 antibody.

Example 4 Affinity Measurement of AR201

Kinetic characterization of the interaction of AR201 versus a commercially available antibody specific for RSV F protein (palivizumab) was conducted by surface plasmon resonance (Biaffin GmbH, Kassel, Germany). AR201 was produced and purified as described above. Recombinant human RSV F protein was covalently immobilized via amine coupling to a CM5 sensor chip for kinetic characterization of the interaction with the antibodies using surface plasmon resonance on a Biacore 2000 instrument (GE Healthcare, Biacore AB, Uppsala, Sweden). For the kinetic analysis of the antibody AR201, dilutions of AR201 antibody were prepared starting at 100 nM and ending at 49 pM after twelve 1:1 dilutions in running buffer. Samples were injected for an association time of two mM, and dissociation was monitored upon switching to running buffer for 30 mM at a flow rate of 30 pL/min. Bound antibody was removed after each injection by surface regeneration using 100 mM HCl (3x 10 sec). Data evaluation was performed by global fitting of the binding curves assuming a Langmuir 1:1 binding model using the Biacore Evaluation software version 4.1. The determined dissociation constant (KD) for AR201 is 58±6 pM. In comparison, palivizumab has a significantly lower KD value of 960 pM (www.ema.europa.eu/docs/en_GB/document_library/EPAR_-Scientific_Discussion/human/000257/WC500056731.pdf). Results are provided in Table 1.

TABLE 1 Association and dissociation constants for AR201. Analyte k_(diss) in s⁻¹ k_(ass) in M⁻¹ s⁻¹ K_(D) in M AR201 (2.7 ± 0.1) × 10⁻⁵ (4.7 ± 0.5) × 10⁵ (5.8 ± 0.6) × 10⁻¹¹

Example 5 Binding of AR201 to RSV Reference Strains

The binding of AR201 to RSV reference strains was tested by ELISA assay. The RSV reference strains RSV-A Long (ATCC VR-26), RSV-B (ATCC VR 1580) and RSV-A2 (ATCC VR-1540) were all purchased from LCG (LGC Standards S.a.r.l., Molsheim, France) and amplified in Vero cells. Subsequently, the supernatant of positive wells was cryopreserved at −80 ° C. until further use. For the ELISA, assay plates were coated with a polyclonal anti-RSV antibody at 500 ng/ml. Meanwhile, 10 μg/ml AR201 or human monoclonal IgG1 isotype control antibody were incubated with RSV at a 1:10 dilution of the frozen amplified RSV strains. After 1 hour, the immune complexes were transferred to the coated ELISA plates and a secondary anti-human IgG-HRP labeled antibody was added. Bound human IgG was detected by colorimetric measurement. As shown in FIG. 7, all three reference strains are recognized by AR201.

Example 6 Neutralization of Reference Strains by AR201

The neutralization of RSV reference strains by AR201 and palivizumab was tested in an infectivity assay. Virus stock (25-250 pfu/well) of the reference strains RSV-A Long (ATCC VR-26) and RSV-B (ATCC VR 1580) was incubated with a two-fold dilution series of antibody solution, and then added to Vero cells that had been seeded in 24-well plates the day before. After four hours, the inoculum was replaced by a semi-solid agarose overlay and incubated for another three days. Subsequently, cells were fixed and stained by immunohistochemistry for the presence of RSV-specific plaques. The percentage of neutralization for each concentration of the RSV-specific antibodies was calculated based on the number of plaques per well. Based on the concentration of the antibody stock, the minimal neutralizing antibody concentration achieving >50% virus neutralization was calculated. As shown in Table 2, AR201 was at least twice as efficient in neutralizing viral particles of either strain and preventing the formation of syncytia indicative of an active, reproductive infection, as compared to palivizumab. An IgG isotype human monoclonal control antibody had no effect on the prevention of infections of Vero cells with either of the RSV reference strains.

TABLE 2 Estimated half-maximal effective antibody concentration (ng/mL). Isotype Ratio Palivizumab AR201 control Palivizumab/AR201 RSV-A/Long 100 ng/ml  40 ng/ml >1000 2.5 RSV-B 200 ng/ml 100 ng/ml >1000 2

Example 7 Binding of AR201 to Clinical Isolates of RSV

The recognition of fresh isolated clinical RSV isolates by AR201 was tested by ELISA assay. Clinical RSV isolates (21 separate isolates) were collected from nasopharyngeal mucus from patients testing positive for RSV. The collected mucus was cultured by incubation on Vero cells, and supernatant of positive wells (as identified by formation of syncytia) were serially passaged twice onto fresh Vero cells. Supernatant of positive wells was cryopreserved at −80 ° C. until further use. For binding analysis, ELISA plates were coated with individual RSV isolates (diluted 1:10), and subsequently incubated with AR201, palivizumab, or a human monoclonal IgG isotype control antibody, at 1 μg/mL. A secondary anti-human IgG-HRP labeled antibody was added to each well. Bound human IgG was detected by colorimetric measurement. The background absorbance, as determined by the signal of the control IgG monoclonal antibody, was deducted twice from the signal detected with AR201 or palivizumab and any absorbance value greater than 0.1 was considered a positive signal. The experiment was repeated three times; a representative experiment is shown in FIGS. 8A and 8B. AR201 recognized all 21 clinical isolates to varying extent, whereas palivizumab always showed a lesser signal intensity. In order to identify palivizumab-resistant strains, the ratio of the binding signal of AR201 over palivizumab was analyzed. Any clinical isolate with a binding signal ratio of 5 or greater of AR201 over palivizumab was considered a palivizumab-resistant isolate.

The results of the analysis are shown in FIG. 8C and Table 3. AR201 recognized all 21 clinical isolates to varying extent, whereas palivizumab always showed a lesser signal intensity. Palivizumab failed to recognize 4 of the 21 isolates, notably the clinical isolates #6, #9, #12 and #20. Clinical isolate #20 had a ratio just above 5.

TABLE 3A Recognition of clinical RSV isolates Monoclonal Antibody Recognition of clinical isolate AR201 21/21 Palivizumab 17/21 Human IgG control mAb  0/21

In further analysis, 68 additional isolates were analyzed by an alternative method: plaque reduction assay. This second assay gave a different output format compared to the first assay (ELISA): for isolate #1 and isolate #20 (“first, ELISA assay” a series of antibody dilutions were tested to find the IC50 which was considered for further testing. In the plaque reduction assay, analysis involved testing ONE antibody concentration and counting how many plaques were formed. Fewer plaques indicated antibody has rendered the virus less infective.

42 RSV/A isolates and 26 RSV/B-isolates were tested for neutralization by AR201 and palivizumab in the plaque reduction assay. A solution of 20 μg/m1 AR201 or palivizumab with 5E4 PFU/ml virus were incubated for 1 hr at 36° C., serially diluted and added to a nearly confluent monolayer of HEp-2 target cells in 24-well plates. After 1.5 hours the supernatant was aspirated, cells were overlayed with media and further incubated. Cytopathogenic effects were monitored daily and plates were fixed and stained after well-defined plaques had formed. Plaque forming units (PFUs) were counted and reduction in PFUs by AR201 or palivizumab was calculated.

In the plaque reduction assay, AR201 reduced the number of plaques in all cases to less than 1%, whereas palivizumab showed such strong reduction only on 45% of isolates. For RSV/A viruses, the ability to reduce plaques was on average 100-fold stronger for AR201 than for palivizumab. For RSV-B isolates, AR201 showed a 20-fold stronger reduction than palivizumab. AR201 fully inhibited plaque formation (sterilizing immunity) for 38 out of 42 tested A-strains (palivizumab: 2 out of 42 A strains), and for 23 out of 26 tested B-strains (palivizumab 7 out of 26 B-strains). Of the 68 tested A- and B-isolates, 59 were more efficiently neutralized by AR201 than by palivizumab. The other 9 were fully neutralized by both antibodies (0 plaques formed)—no difference in neutralization efficiency at the tested antibody concentration could be determined.

TABLE 3B Summary of the Effect of AR201-Compared to Palivizumab Monoclonal Antibodies on Multiple RSV/A and RSV/B Strains as Determined by a Plaque Reduction Assay. Limit of detection 0.5 PFU (−0.3 Log₁₀); negative samples without plaques were given half the value of the detection limit (0.25 PFU (−0.6Log₁₀)). P value^(a) Log₁₀ PFU/0.2 mL Pmab v Virus Control Palivizumab AR201 AR201 RSV/A (n = 42) 3.49 1.58 −0.42 <0.001 Mean SD 0.34 0.80 0.61 Median 3.48 1.60 −0.60 RSV/B (n = 26) 3.40 0.79 −0.52 <0.01 Mean SD 0.36 1.03 0.35 Median 3.40 1.18 −0.60 ^(a)Kruskal-Wallis nonparametric test, two-tailed. Pmab, palivizumab.

TABLE 3C Effect of AR201 and Palivizumab on ≧100-fold Reduction in PFU and Sterilizing Immunity with Strains of RSV/A and RSV/B Viruses Virus >100-Fold Reduction Sterilizing Immunity^(a) Strain Palivizumab AR201 P value^(b) Palivizumab AR201 P value² RSV/ 13^(c) 42 <0.0001 2 38 <0.0001 A (n = 42) RSV/ 18   26 0.011 7 23 <0.0001 B (n = 26) ^(a)Sterilizing immunity is defined as no plaques visible in the undiluted sample. For statistical purposes no plaques were assigned a value of 0.25 PFU. ^(b)Fisher's Exact Test, two-sided. ^(c)Number of isolates evaluated.

Example 8 Neutralization of Clinical Isolates by AR201

The neutralization of two clinical RSV isolates (isolate #1 and #20) by AR201 and palivizumab was tested in an infectivity assay. The two RSV isolates were amplified on Vero cells and supernatant cryopreserved until further use. The dilution of the stock solution achieving a 50% infectivity rate in a tissue culture (TCID) was calculated based on the Spearman and Karber algorithm (Hierholzer et al., 1996, in Virology Methods Manual, edited by BMJ Mahy and HO Kangro, London: Academic Press, p.47). Virus stock were thawed and diluted to 10 x TCID50, and incubated with serial dilutions of antibodies AR201, palivizumab, or IgG1 isotype human control monoclonal antibody. The mixture was added to confluent Hep2 cells (ATCC CCL-23) in 96-well plates and incubated for four days. Cells were fixed and infected cells were detected with an anti-RSV mouse monoclonal antibody clone 631 (Milan Analytica AG, Switzerland). The rate of infection and IC50 values were calculated based on colorimetric measurement of the bound antibody by applying a variable slope-four parameter equation (GraphPad Prism Software V5.02, GraphPad Software Inc. San Diego, Calif., US). The IC50 served as measure of the neutralization activity. IC50 is the antibody concentration (g/mL) that neutralizes the infectivity capacity of an RSV infective dose by 50%.

Results are shown in Table 4. AR201 had an IC50 that was 18 times lower than palivizumab for isolate #1. For isolate #20, no neutralization capacity was observed for palivizumab, confirming that palivizumab does not bind to clinical isolate #20, whereas AR201 showed a similar IC50 on isolate #20 as seen with isolate #1.

TABLE 4 IC50 of AR201 and palivizumab for clinical isolates #1 and #20. IC50 microM RSV-Isolate 1 RSV-Isolate 20 AR201 1.702 1.706 Palivizumab 30.601 >1000 IgG1 Isotype control >1000 >1000 Ratio palivizumab/AR201 18.0 n/a

Example 9 Sequencing of F Protein Sequence of Palivizumab-Resistant Clinical Isolate #20

The nucleotide and amino acid sequences of the F protein of clinical isolate #20 were determined. RNA was isolated from amplified clinical isolate #20 and cDNA was generated with F protein specific primers (Primer RSV-A F protein: GAAATTAAACCTGGGGCAAATAACC [SEQ ID NO.: 19]; Primer RSV-B F protein: ACAAAATMAACTCTGGGGCAAATAAC [SEQ ID NO.: 20]). The resulting fragment (expected size: 1935 bp) was amplified using specific primers (RSV-7607: CTTCGYGACATATTTGCCCCAG [SEQ ID NO.: 21]; ZhaoF: GGGGCAMTMCMTGGAGTTGC [SEQ ID NO.: 22]). Amplified DNA was sequenced by Sanger sequencing at Microsynth (Balgach, Switzerland).

The nucleotide sequence of the coding region for the F protein of clinical isolate #20 (SEQ ID NO.: 17) is provided in FIG. 9A. The corresponding amino acid sequence (SEQ ID NO.: 18) is provided in FIG. 9B.

Comparison to the published sequence of the postulated epitope region of palivizumab (McLellan J S. et al., 2010), identified as the sequence of amino acids 254 to 277 of the RSV F protein A2 strain, revealed a mutation of lysine at the position 272 to asparagine (K272N) for the clinical isolate #20, as shown in FIG. 10. A mutation at position 272 from lysine to another amino acid was described earlier as critical for loss of binding of palivizumab to the F protein (Zhu, et al., 2011; Adams et al., 2010), confirming the inability of palivizumab to bind within the epitope region of clinical isolate #20, confirming the sequence of isolate #20 region does not include an amino acid sequence functional as a palivizumab epitope. Further, two palivizumab-resistant mutant viruses were generated and both had a single amino acid (aa) mutation at position 262.

Example 10 Generation of Palivizumab Resistant Mutants

Palivizumab-resistant mutants of RSV/A/Tracy and RSV/B/18537, respectively were generated after serial passage of the viruses in increasing concentrations (40, 80, 160 and 320 μg/mL) of palivizumab. Two palivizumab-resistant mutant viruses were isolated and both had a single amino acid (aa) mutation at position 262 as shown in FIG. 10. Both parent strains had asparagine (an amide, neutral polar aa) at aa262 and both palivizumab resistant mutants had tyrosine (an aromatic neutral polar aa). The N262Y change occurred in the putative antigenic II domain and within the palivizumab defined epitope region (aa262-276). Most of the published palivizumab resistant mutants have changes that occur in aa 272, 268, 275 and 276. As shown in Table 5 below, the palivizumab resistant mutant of RSV/A/Tracy or RSV/B/18537 infected cotton rats equally well compared to the parent virus, however, the mutant virus was resistant to the effects of palivizumab immunoprophylaxis.

TABLE 5 Comparison of the Inhibitory Effect of AR201 and Palivizumab (40 μg/ mL each) on Palivizumab-resistant RSV/A/Tracy and RSV/B/18537. Log₁₀ Virus Titer (log₁₀ PFU/0.2 mL) Reduction v Control AR201E- AR201E- PR-RSV Control Palivizumab N Palivizumab N RSV/A/ 3.80 3.61 3.18 0.19 0.63 Tracy (n = 1) RSV/B/ 3.36 3.31 2.06 0.05 1.30 18537 (n = 1)

Example 11 Cross Inhibition on RSV Clinical Isolates with AR201 and Palivizumab

Palivizumab is the only anti-RSV monoclonal antibody that has been approved by the FDA to date for the prevention of RSV infection in infants and neonates. Palivizumab binds to antigenic region A on the F protein of RSV and inhibits fusion events of RSV with human lung epithelial cells.

Efficient cross-inhibition of binding of two independent monoclonal antibodies can only occur if both antibodies recognize an epitope, or epitopes in close proximity (e.g., same epitope region), such that one antibody when bound to its epitope exerts steric hindrance for binding of the other antibody.

Intact RSV particles were pre-incubated with competitor antibody (palivizumab, AR201, or control at 20 μg/mL) for one hour and subsequently added to polystyrene ELISA plates that had been coated with either palivizumab or AR201. After incubation for one hour at 4 ° C., plates were washed and polyclonal rabbit-anti-RSV-HRP labeled antisera was added to each well. Bound viral particles were detected by colorimetric measurement. Maximum binding of RSV to the immobilized antibody in the presence of the competitor antibody was determined relative to binding in the presence of the monoclonal human IgG control antibody.

Results are provided in Table 6. Pre-incubation of RSV viral particles with AR201 reduced binding to AR201 coated plates to 22.5%, whereas pre-incubation with palivizumab affected binding to AR201 coated plates less and reduced the signal to only 29.2% of the control value. Vice versa, pre-incubation of RSV viral particles with AR201 reduced binding of RSV to palivizumab-coated plates to 40.8%, whereas pre-incubation with palivizumab reduced binding to palivizumab-coated plates at a slightly lower level, 35.3%.

These results indicate that AR201 targets the identical antigenic region on RSV F protein as palivizumab. That is, AR201 and palivizumab may have target epitopes in the similar region of amino acids on the RSV F-protein, but these antibodies may not necessarily interact with the same amino acid epitope members within the region. The difference in cross-inhibition indicates that AR201 recognizes an epitope similar to or close to the epitope of palivizumab, but not the identical epitope.

TABLE 6 Cross-inhibition of AR201 or Palivizumab for Binding of RSV to Antibody-Coated Plates Coating mAb Inhibiting mAb palivizumab AR201 AR201 40.8% 22.5% palivizumab 35.3% 29.2% control IgG mAb 100.0% 100.0%

Example 12 Protease Digestion of F Protein

Asp-N is an endoprotease which selectively cleaves peptide bonds N-terminal to aspartic acid residues. Asp-N digestion of the RSV F protein will cut the postulated palivizumab epitope N-terminal of amino acid positions 263 and 269, generating two protein fragments of 62 amino acids and 40 amino acids in length. Each peptide contains only a portion of the intact epitope for palivizumab, as shown in FIG. 11.

Purified recombinant F protein was incubated for 5 hours with Asp-N (Promega, Madison, Wis., US) at 37 ° C., according to manufacturer's instructions, in the presence of dithiothreitol (DTT) and iodacetamide, and subsequently coated onto ELISA plates. Antibody (AR201 or palivizumab) was added to the plate at two different concentrations, followed by addition of a polyclonal anti-human IgG-HRP labeled antibody. Bound antibody was detected based on colorimetric measurement.

Palivizumab did not recognize any of the fragments of the complete digest of the RSV F protein, whereas AR201 still bound to as shown in FIG. 10. This demonstrates that the two antibodies do not share the same epitope, and AR201 recognizes a combination of epitope amino acids in the 254-277 epitope region of the RSV F protein different from the amino acids making up the palivizumab target epitope amino acids. Furthermore, the epitope binding region exists in a three dimensional configuration such that AR201 binds differently than palivizumab.

Example 13 Conformational Epitope Mapping

To further characterize the epitope of AR201, permutations of peptides containing the epitope region sequence were 3-dimensonally structured to mimic non-linear epitopes and tested for binding of AR201 and palivizumab by ELISAs (CLIPS technology: Timmerman et al, 2007 Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology. J. Mol. Recognit. 20:283-99; Slootstra et al 1996; Structural aspects of antibody-antigen interaction revealed through small random peptide libraries, Molecular Diversity 1:87-96; conducted at Pepscan Presto BV, Netherlands; see below). The main goal of this study was to determine if AR201 and palivizumab bind the same/different epitopes.

The segment of interest was narrowed down to NSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSI, a segment of the F-Protein from RS-Virus (amino acids 254-288). A set of CLIPS structured peptides, all covering this segment, were designed, synthesized in the pepscan array format and screened in pepscan ELISA.

To summarize the first phase of the study, it was found that both antibodies seemed to bind within overlapping epitope regions in the segment investigated. However, the binding specificity was very different, meaning that the CLIPS seem to put the peptides in a conformation favoring one antibody, while the other favors the second.

Based on the result a detailed follow-up alanine-scan of two identified binders was conducted. For both antibodies similar positions were found to be essential (in bold and underlined):

NSE LL SL IN DMP ITND Q KK LMS S NVQIVRQQSYSI [SEQ ID No: 29]

Remarkably, with AR201 various single or double alanine mutations increased binding activity, especially in the NVQIVRQQSYSI part of the peptide (which is not the “essential part” of the epitope recognized by palivizumab). A similar result was observed with lengthening the peptide with MSI in the first part of the study.

The overall conclusion is that both antibodies bind to NSELLSLINDMPITN DQKKLMSSN (SEQ ID NO: 29) but with different specificity. Small structural differences of the epitope, but also outside the epitope, affect the binding of both antibodies in completely different ways. Again, this suggests the binding interaction and epitope amino acids are different between AR201 and palivizumab.

Example 14 Production of AR201 in plants

AR201 was produced in the plant Nicotiana benthamiana using the magnifection technology (Giritch et al, 2006 , Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proceedings of the National Academy of Sciences of the United States of America 103(40):14701-14706), which allows the production of antibodies within a very short timeframe. The use of plants with diminished fucosyl- and xylosyl-transferase (Strasser et al, 2008, Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnology Journal 6(4):392-402.) enabled the production of antibodies carrying glycans lacking fucose and xylose. Provision of the IgG antibodies without fucose have an increased ability to elicit ADCC (antibody-dependent cell-mediated cytotoxicity) due to their higher affinity for FcRIIIA (CD16a).

For plant growth conditions, vectors, infiltration, extraction and purification, methods outlined elsewhere were followed (Castilho et al, 2011; Rapid high yield production of different glycoforms of Ebola virus monoclonal antibody. PloS one, 6(10), e26040, Giritch et al, 2006). In brief, codon-optimized heavy and light chains were cloned into plant expression vectors (Giritch et al, 2006) and transformed into Agrobacterium tumefaciens. Equal volumes of Agrobacterium cultures grown overnight were mixed in infiltration buffer (1 mM MES/10 mM MgSO4 pH 5.5) resulting in a 1:1000 dilution for each individual culture. N. benthamiana plants grown for 4-5 weeks at 22° C. with a 16/8 hr light/dark cycle were inverted and aerial parts were submerged in the bacterial/buffer solution. A vacuum of 0.5 bars was applied for 2 min and upon release the leaves got infiltrated with the bacterial solution. Plants were returned to the growth room and leaf tissue was extracted 7 days later in a blender with 250 ml chilled buffer (100 mM Tris, 1 mM EDTA, 40 mM ascorbic acid) per kg leaf material. The extract was clarified by lowering the pH to 4.8 with 1 M phosphoric acid then re-adjusting to pH 7.5 with 2 M Tris base to insolubilize plant polymers, by centrifugation (16,000 g, 30 mM) and by filtration (0.2 μm). Purification on a protein A resin was followed by polishing via anion chromatography. Upon TFF concentration the extract is filtered (0.2_(l)am) and immediately applied to a Protein A column. Upon washing of the column (50 mM HEPES/100 mM NaCl, pH 7.5), elution (0.1 M acetic acid, pH 3.0) neutralization of the eluate (2 M Tris, pH 8.0) and addition of Tween 80 to 0.01%, the mAb solution was polished via Q filtration (Mustang Acrodisc Q membrane; Pall), aliquoted and stored at −80° C. until use.

Antibody AR201 produced by CHO cells and alternatively in plants were compared with palivizumab in a Cotton Rat Model, as discussed below. Furthermore 2 AR201 mutants were produced in plants in order to increase the half-life of the antibody in serum. AR201-YTE is a triple mutation M252Y/S254T/T256E (YTE) which was introduced into the Fc portion. Similarly, the Fc portion can be modified to present neonatal Fc receptor for IgG (FcRn) to enhance half-life.

Cotton rats (A-F) were treated with 5 mg/kg intramuscular AR201, AR201-YTE, AR201-XTND and palivizumab, respectively and challenged with RSV/A/Tracy on day 0.

Below it is shown that material from CHO cells and material from plants is comparable in the Lung Lavage Plaques Reduction Assay and in the Neutralization Assay. Further, the mutants generated for AR201 show comparable results to AR201 in the 2 assays mentioned above. Note “nic” indicates Nicotiana expression and “CHO” indicates CHO cell expression. Following letters, —XTND or —YTE, indicate AR201 antibody mutants.

Comparison CHO/Plant Material: RSV/A/Tracy Titers in Lung Lavage Plaque Reduction Assay

TABLE 7 RSV/A/Tracy titers in lung lavage fluids on day +4 RSV titer (log₁₀ PFU/g lung) in cotton rat Change T test/2 Group Treatment A B C D E F Mean SD (log₁₀) v. Gp 1* 1 Untreated 5.57 5.33 5.22 5.43 5.19 5.19 5.32 0.15 — — 2 Nic-AR201 2.69 2.43 3.05 2.50 3.01 3.16 2.81 0.31 −2.52 <0.0001 3 Nic-AR201-XTND 2.51 2.50 2.43 2.24 3.52 3.30 2.75 0.53 −2.57 <0.0001 4 CHO-AR201 1.43 2.64 3.09 2.80 1.67 1.71 2.22 0.70 −3.10 <0.0001 5 CHO-AR201-XTND 2.80 3.10 2.49 3.29 2.41 2.76 2.81 0.34 −2.52 <0.0001 6 Palivizumab 3.47 3.30 3.21 2.77 4.30 4.09 3.52 0.57 −1.80 <0.0001 *Minimum detection ~1.4 log₁₀/g lung. Additional significant P values (Student t test, two-tailed): Group 6 v 2, 3, 4, 5, P ≦ 0.035.

RSV/A/Tracy Serum Neutralizing Antibody Titers:

TABLE 8 RSV/A/Tracy serum neutralization titer on Day 0 RSV/A neutralization titer (log₂) in cotton rat T test/2 v. Group Treatment A B C D E F Mean SD Gp 1* 1 Untreated 2 2 2 2 2 2 2 0 — 2 Nic-AR201 9.0 8.5 9.0 8.5 8.5 8.5 8.7 0.3 <0.00001 3 Nic-AR201-XTND 8.5 8.0 8.5 8.0 8.5 8.5 8.3 0.3 <0.00001 4 CHO-AR201 9.0 8.5 9.0 9.5 8.5 9.5 9.0 0.4 <0.00001 5 CHO-AR201-XTND 9.5 8.5 8.5 8.0 8.5 9.0 8.7 0.5 <0.00001 6 Palivizumab 6.0 5.0 5.5 5.5 5.0 5.5 5.4 0.4 <0.00001 *Student t test, two-tailed. Minimal detection = 2.5; for statistical analysis a value of <2.5 was counted as 2. Additional significant P values (Student t test, two-tailed): Group 3 v 2, 4, P ≦ 0.049; Group 6 v 2, 3, 4, 5, P < 0.00001.

TABLE 9 RSV/A/Tracy serum neutralization titer on Day +4 RSV/A neutralization titer (log₂) in cotton rat T test/2 v. Group Treatment A B C D E F Mean SD Gp 1* 1 Untreated 2 2 2 2 2 2 2 0 — 2 Nic-AR201 9.0 8.0 8.0 9.0 8.5 7.5 8.3 0.6 <0.00001 3 Nic-AR201-XTND 8.0 8.5 8.0 8.0 7.5 8.0 8.0 0.3 <0.00001 4 CHO-AR201-N 8.5 8.0 8.5 9.0 9.0 9.0 8.7 0.4 <0.00001 5 CHO-AR201-XTND 8.5 9.0 9.0 8.0 8.5 8.5 8.6 0.4 <0.00001 6 Palivizumab 5.5 5.5 5.0 5.0 5.5 5.5 5.3 0.3 <0.00001 *Student t test, two-tailed. Minimal detection = 2.5; for statistical analysis a value of <2.5 was counted as 2. Additional significant P values (Student t test, two-tailed): Group 3 v 4, 5, P ≦ 0.016; Group 6 v 2, 3, 4, 5, P < 0.00001.

The mutants generated for AR201 show comparable results to AR201 in RSV/A/Tracy Lung Lavage Plaque Reduction Titer Assay:

TABLE 10 RSV/A/Tracy titers in lung lavage fluids on day +4 RSV titer (log₁₀ PFU/g lung) in cotton rat Change T test/2 v. Group Treatment A B C D E F Mean SD (log₁₀) Gp 1* 1 Untreated 5.57 5.40 5.40 5.43 5.67 5.44 5.48 0.11 — — 2 AR201 3.09 3.37 2.94 2.82 2.65 1.71 2.76 0.57 −2.72 <0.00001 3 AR201-YTE 3.24 2.94 3.32 2.52 4.27 2.61 3.15 0.64 −2.33 <0.00001 4 AR201-XTND 2.57 3.08 2.43 2.55 2.38 2.48 2.58 0.26 −2.90 <0.00001 5 Palivizumab 3.63 3.32 3.44 3.76 4.20 3.48 3.64 0.32 −1.85 <0.00001 *Minimum detection = 1.3 log₁₀/g lung. Additional significant P values (Student t test, two-tailed): Group 5 v 2, 4, P ≦ 0.0082.

RSV/A/Tracy Serum Neutralizing Antibody Titers

TABLE 11 RSV/A/Tracy serum neutralization titer on Day 0 RSV/A neutralization titer (log₂) in cotton rat T test/2 v. Group Treatment A B C D E F Mean SD Gp 1* 1 Untreated 2 2 2 2 2 2 2 0 — 2 AR201 8.0 7.0 7.5 7.5 8.0 7.5 7.6 0.4 <0.00001 3 AR201-YTE 7.0 7.5 7.0 8.0 7.0 7.5 7.3 0.4 <0.00001 4 AR201-XTND 7.5 7.5 8.0 8.0 7.0 7.0 7.5 0.4 <0.00001 5 Palivizumab 5.5 5.0 5.0 5.0 5.0 4.5 5.0 0.3 <0.00001 *Student t test, two-tailed. Minimal detection = 2.5; for statistical analysis a value of <2.5 was counted as 2. Additional significant P values (Student t test, two-tailed): Group 5 v 2, 3, 4, P < 0.00001.

TABLE 12 RSV/A/Tracy serum neutralization titer on Day +4 RSV/A neutralization titer (log₂) in cotton rat T test/2 Group Treatment A B C D E F Mean SD v. Gp 1* 1 Untreated 2 2 2 2 2 2 2 0 — 2 AR201 6.5 6.5 6.5 6.5 7.5 6.5 6.7 0.4 <0.00001 3 AR201-YTE 6.0 6.5 6.5 7.0 5.5 7.0 6.4 0.6 <0.00001 4 AR201-XTND 6.0 6.5 7.5 7.0 7.0 7.0 6.8 0.5 <0.00001 5 Palivizumab 4.5 4.5 4.0 5.0 4.0 4.0 4.3 0.4 <0.00001 *Student t test, two-tailed. Minimal detection = 2.5; for statistical analysis a value of <2.5 was counted as 2. Additional significant P values (Student t test, two-tailed): Group 5 v 2, 3, 4, P ≦ 0.000031.

It is an optional aspect of the invention that the antibody (e.g., AR201) comprise either or both of modified glycosylation (e.g., plant expression) and Fc mutations. We have the expectation that a combination of plant expression (e.g., antibodies presenting glycans lacking fucose and/or xylose) and Fc mutations (M252Y/S254T/T256E-YTE) will either additively or synergistically enhance the effectiveness of the antibody in preventing and treating RSV infections.

Example 15 Effectiveness of IM Administration of AR201 and Palivizumab in the RSV/A/Tracy Cotton Rat Model

Microneutralization assays were performed to test for neutralizing antibodies to RSV/A/Tracy in the serum of cotton rats. Serum samples were heat inactivated at 56° C. for 30 min, serially two-fold diluted in duplicates and mixed with plaque purified RSV/A/Tracy (RSV/A). 96-well microtiter plates with nearly confluent HEp-2 cells were infected, further incubated and plaque formation was evaluated as described above (Example 8, “Plaque reduction assay”). The neutralizing antibody titer was defined as the serum dilution at which >50% reduction in plaque formation was observed.

The effectiveness of IM administration of AR201 and Palivizumab to reduce RSV titers was tested in a cotton rat model. Cotton rats (A-F) received 5 mg antibody per kg bodyweight (or control formulation) on day -1 and were challenged with RSV/A/Tracy with 1.35×10⁵ plaque forming units per cotton rat on day 0. Directly upon challenge blood was collected for determination of RSV-neutralizing antibody levels. On day +4 animals were sacrificed, blood samples were taken and the left lung lobe was removed. Blood was removed from lobe and it was transpleurally lavaged using 3 mL of Iscove's media with 15% glycerin mixed with 2% FBS-MEM (1:1, v:v). Lavage fluid was recovered by gently pressing the inflated lobe and used to lavage the right lobe following the same technique. RSV titers were determined as described above (example 8, “Plaque reduction assay).

Cotton rats that had received a prophylactic dose of AR201 had a substantially higher titer of RSV/A/Tracy neutralizing antibodies in the serum than cotton rats that had received palivizumab. Although AR201 and palivizumab were dosed at 5 mg/kg, at the time of RSV/A/Tracy challenge (day 0) serum neutralizing antibody titer for AR201 was significantly higher than for palivizumab. This difference in titer of neutralizing antibodies was reflected by the reduced titer of virus on day 4 in lung lavage fluid of AR201 treated cotton rats (see below).

Both, AR201 and palivizumab reduced the virus titer in lung lavage fluid, however, while palivizumab reduced the titer only by a factor of ˜70, AR201 reduced the lung virus titer over 500-fold and was thus several fold as effective as palivizumab. Therefore, AR201 is a more potent monoclonal antibody in reducing lung virus titer compared to palivizumab in the RSV/A cotton rat model. See FIG. 13.

TABLE 13 RSV/A/Tracy serum neutralization titer on Day 0 RSV/A neutralization titer (log₂) in cotton rat Group Treatment A B C D E F Mean SD T test/2 v. Gp 1* 1 Untreated 2 2 2 2 2 2 2 0 — 2 AR201 8.0 7.0 7.5 7.5 8.0 7.5 7.6 0.4 <0.00001 3 Palivizumab 5.5 5.0 5.0 5.0 5.0 4.5 5.0 0.3 <0.00001 *Student t test, two-tailed. Minimal detection = 2.5; for statistical analysis a value of <2.5 was counted as 2. Additional significant P value (Student t test, two-tailed): Group 2 v 3, P < 0.00001.

TABLE 14 RSV/A/Tracy serum neutralization titer on Day +4 RSV/A neutralization titer (log₂) in cotton rat Group Treatment A B C D E F Mean SD T test/2 v. Gp 1* 1 Untreated 2 2 2 2 2 2 2 0 — 2 AR201 6.5 6.5 6.5 6.5 7.5 6.5 6.7 0.4 <0.00001 3 Palivizumab 4.5 4.5 4.0 5.0 4.0 4.0 4.3 0.4 <0.00001 *Student t test, two-tailed. Minimal detection = 2.5; for statistical analysis a value of <2.5 was counted as 2. Additional significant P values (Student t test, two-tailed): Group 2 v 3 P ≦ 0.000031.

TABLE 15 RSV/A/Tracy titers in lung lavage fluids on day +4 RSV titer (log₁₀ PFU/g lung) in cotton rat Change T test/2 v. Group Treatment A B C D E F Mean SD (log₁₀) Gp 1* 1 Untreated 5.57 5.40 5.40 5.43 5.67 5.44 5.48 0.11 — — 2 AR201 3.09 3.37 2.94 2.82 2.65 1.71 2.76 0.57 −2.72 <0.00001 3 Palivizumab 3.63 3.32 3.44 3.76 4.20 3.48 3.64 0.32 −1.85 <0.00001 *Minimum detection = 1.3 log₁₀/g lung. Additional significant P values (Student t test, two-tailed): Group 2 v 3.

Example 16 Enhanced AR201

Different IgG mutations have been described to prolong the half-life of an IgG in serum by changing its binding to the neonatal Fc Receptor (FcRn). FcRn binds IgGs at acidic pH in the endosomes and releases it at neutral pH at the cell. Mutations of the IgG on the FcRn binding site, but also at more distant positions can influence this binding behavior, increase the affinity of the antibody for FcRn, and prolong the half-life of an IgG roughly 3-4 fold.

Two mutants were generated AR201-YTE and AR201-XTND (see Example 14, above) in order to produce a high affinity RSV-antibody with a prolonged half-life. This will allow reduced dosing frequency of these antibodies from roughly once per month to only once per RSV infection season (4-6 months). Especially for high-risk infants, their parents and their physicians this will be a significant improvement over the current treatment/prophylaxis options.

Antibody effector functions like antibody dependent cell-mediated cytotoxicity (ADCC) in fighting viral infections is important. ADCC is mediated by antibodies that crosslink antigen and immune cells e.g., natural killer cells, by binding at the same time to the epitope and to FcRIIIA (CD16a), the “ADCC-receptor”, which is expressed on certain immune cells. This elicits ADCC, or killing of the target cells. Higher affinity of antibody to the Fc₇RIIIa translates to increased killing of the target by the immune cells, i.e. to better ADCC. The affinity of the antibody for the receptor can be influenced by altering the amino acid sequence of the antibody, but also by altering the glycosylation profile of the antibody (Shinkawa, J. Biol. Chem. 2003; 278(5): 3466-3473). Absence of a specific glycan moiety (α-1,6-fucose) drastically increases affinity for the FcRIIIa receptor and thereby also ADCC). Unfortunately, most of the commonly used IgG production systems produce antibodies with a high degree of fucosylation (˜95%). Alternatives, allowing the production of non- or low-fucosylated antibodies include plant production systems, discussed above.

Production of AR201 in a plant-based antibody production system (e.g., Nicotiana benthamiana with the Magnifection system) resulted in production of AR201 with hardly detectable fucose. The production of antibodies (e.g., AR201-YTE) with Fc modifications has shown good affinity and will enhance antibody half-life. An improved product can be an antibody with both of these features. For example, an ant-RSV antibody (such as AR201) can be designed with Fc mutations for better half-life, and expressed in plants for better cytotoxicity. The combination can provide the efficacy beyond the individual contributions alone. Such an AR201, in the works, is an antibody which combines both effects of longer half-life by amino acid mutations as well as increased ADCC by optimized glycosylation profiles and/or amino acid mutations, and is expected to by superior to any anti-RSV described so far. Such improved anti-RSV antibodies can provide lifesaving advantages for infants, the elderly, and immunocompromised patients.

All of the references described herein are incorporated by reference in their entireties. 

1 to 32 (canceled)
 33. A cell comprising a first cDNA comprising a sequence which encodes a heavy chain variable region and a second cDNA comprising sequence which encodes a light chain variable region, wherein the cell produces an antibody construct or antibody fragment comprising the heavy chain variable region and the light chain variable region, wherein the heavy chain variable region encoded by the first cDNA sequence comprises (a) a heavy chain complementarity determining region (CDR) CDR1 comprising the amino acid sequence GASINLYD (SEQ ID NO.: 8), (b) a heavy chain CDR2 comprising the amino acid sequence GYISGST (SEQ ID NO.: 9), and (c) a heavy chain CDR3 comprising the amino acid sequence ARDVGWGPQYYYGLDV (SEQ ID NO.: 10); wherein the light chain variable region encoded by the second cDNA sequence comprises (a) a light chain CDR1 comprising the amino acid sequence HSVQSTS (SEQ ID NO.: 14), (b) a light chain CDR2 comprising the amino acid sequence GGS (SEQ ID NO.: 15), and (c) a light chain CDR3 comprising the amino acid sequence QQSDRSPPIT (SEQ ID NO.: 16); and, wherein the cell is other than a beta lymphocyte.
 34. The cell of claim 33, wherein the cell is selected from the group consisting of: plant cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells.
 35. The cell of claim 33, wherein the antibody construct or antibody fragment further comprises a non-human N-glycan structure.
 36. The cell of claim 33, wherein the light chain and heavy chain CDR sequences originate from different beta lymphocytes.
 37. The cell of claim 33, wherein the antibody construct or antibody fragment binds with specificity to RSV F protein antigenic region II/A with an affinity of greater than 1×10⁻⁹ M.
 38. The cell of claim 33, wherein the antibody construct or antibody fragment is an IgG₁.
 39. The cell of claim 33, wherein the cell comprises one or more expression vectors, wherein the one or more expression vectors comprise the first cDNA sequence or the second cDNA sequence.
 40. The cell of claim 33, wherein at least one expression vector is stably transfected into the cell and adapted to provide expression of the first cDNA sequence or the second cDNA sequence.
 41. The cell of claim 33, wherein the heavy chain variable region cDNA sequence comprises the nucleotide sequence of SEQ ID NO: 2 or the light chain variable region cDNA sequence comprises the nucleotide sequence of SEQ ID NO:
 4. 42. The cell of claim 33, wherein the light chain and heavy chain are from different antibodies produced by an RSV patient.
 43. The cell of claim 33, wherein the antibody construct or antibody fragment neutralizes at least one RSV strain that is resistant to palivizumab.
 44. The cell of claim 33, wherein the antibody or antibody fragment comprises glycans lacking fucose and xylose; or, wherein the antibody or antibody fragment comprises a Fc region adapted to bind to neonatal receptor FcRn of epithelial cells with at least 10-fold less affinity than AR201 binds the receptor.
 45. An antibody or antibody fragment comprising at least a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (a) a heavy chain complementarity determining region (CDR) CDR1 comprising the amino acid sequence GASINLYD (SEQ ID NO.: 8), (b) a heavy chain CDR2 comprising the amino acid sequence GYISGST (SEQ ID NO.: 9), and (c) a heavy chain CDR3 comprising the amino acid sequence ARDVGWGPQYYYGLDV (SEQ ID NO.: 10); and wherein the light chain variable region comprises (a) a light chain CDR1 comprising the amino acid sequence HSVQSTS (SEQ ID NO.: 14), (b) a light chain CDR2 comprising the amino acid sequence GGS (SEQ ID NO.: 15), and (c) a light chain CDR3 comprising the amino acid sequence QQSDRSPPIT (SEQ ID NO.: 16), wherein the antibody or antibody fragment comprises glycans lacking fucose and xylose.
 46. The antibody or antibody fragment of claim 45, wherein the glycans are non-human N-glycans.
 47. The antibody or antibody fragment of claim 45, wherein the antibody or antibody fragment comprises a Fc region adapted to bind to neonatal receptor FcRn of epithelial cells with at least 10-fold less affinity than AR201 binds the receptor.
 48. The antibody or antibody fragment of claim 45, wherein the antibody construct or antibody fragment recognizes an epitope in a region of SEQ ID NO.: 23 or SEQ ID NO.:
 24. 49. A vector comprising a first nucleic acid comprising a sequence which encodes a heavy chain variable region and a second nucleic acid comprising a sequence which encodes a light chain variable region, wherein the cell produces an antibody construct or antibody fragment comprising the heavy chain variable region and the light chain variable region, wherein the heavy chain variable region encoded by the first nucleic acid sequence comprises (a) a heavy chain complementarity determining region (CDR) CDR1 comprising the amino acid sequence GASINLYD (SEQ ID NO.: 8), (b) a heavy chain CDR2 comprising the amino acid sequence GYISGST (SEQ ID NO.: 9), and (c) a heavy chain CDR3 comprising the amino acid sequence ARDVGWGPQYYYGLDV (SEQ ID NO.: 10); and wherein the light chain variable region encoded by the second nucleic acid sequence comprises (a) a light chain CDR1 comprising the amino acid sequence HSVQSTS (SEQ ID NO.: 14), (b) a light chain CDR2 comprising the amino acid sequence GGS (SEQ ID NO.: 15), and (c) a light chain CDR3 comprising the amino acid sequence QQSDRSPPIT (SEQ ID NO.: 16).
 50. The vector of claim 49, wherein the first nucleic acid or second nucleic acid is a cDNA or wherein the light chain and heavy chain CDR sequences originate from different beta lymphocytes. 